We have identified unusual deletion and insertion polymorphisms that are found with uniform consistency over time in HIV-1 from 8 of 10 long-term non-progressors (LTNP). Two of these individuals appear to have been infected with viruses bearing large, non-revertible deletions in nef. Smaller deletions were noted to cluster in the gag, env and nef genes of other LTNP. We hypothesize that these polymorphisms attenuate the pathogenic potential of the infecting strain. With the proposed studies, we will complete the sequence analysis of the viral genomes from two additional LTNP (one of whom is infected with a virus that is extremely growth-impaired) in order to identify unique, difficult-to-revert gene polymorphisms. We will characterize the effects of the polymorphisms in env and nef on viral replication and gene function. Cell culture systems will be used to compare the growth and cytopathicity properties of recombinant infectious molecular clones of HIV-1 that differ only in the sequences of interest. Transient expression assays, cell culture assays, and biochemical tests will be used to analyze the functional consequences of these naturally occurring polymorphisms. We will also study the potential role of cell-mediated immune responses in the selection and maintenance of these polymorphisms. These detailed analyses should provide unique information regarding the functional consequences of viral gene polymorphisms observed in association with non-progressive infection, and should provide improved understanding of the potential mechanisms for their selection and maintenance. These studies will define the relative importance and functional contribution of individual genetic elements of HIV-1 for pathogenesis in humans, and may also contribute to the development of new strategies to control or prevent HIV-1 infection.